Carmabinoid receptors are molecular targets for marijuana, the most widespread illegal drug of abuse in Western societies. They are densely expressed in areas of the central nervous system (CNS) that participate in the control of appetite, pain, movement and memory. Such functions are strongly affected by cannabinoid drugs, with consequences that include appetite stimulation, analgesia, euphoria, and memory impairment. Although the pharmacology of cannabinoid drugs is now fairly well understood, the endogenous signaling system by which cannabinoid receptors are normally engaged remains largely unexplored. Endogenous ligands for cannabinoid receptors, such as anandamide and 2-arachidonylglycerol (2-AG), have been described. Anandamide is produced in and released from neurons on depolarization, and undergoes a rapid process of biological inactivation consisting of transport into cells and intracellular hydrolysis. Anandamide transport is mediated by a high-affinity facilitated carrier system, the molecular structure of which remains unknown, while anandamide hydrolysis is catalyzed by the enzyme fatty acid arnide hydrolase (FAAH). The first aim of the proposed research is to investigate the mechanisms responsible for anandamide transport. Experiments conducted during the current funding period have revealed several general properties of anandamide transport. The proposed studies will extend these investigations, focusing on the role played by FAAH in driving anandamide internalization. The second aim of our proposed research is to determine what the structural determinants are for anandamide transport. Our previous investigations have led to the discovery of novel inhibitors of anandamide transport. We will further explore the structure- activity relationship of this process by designing and synthesizing novel reverse-amide analogs of anandamide and testing them for the ability to act as selective transport inhibitors. The third aim of our proposal is to develop potent and selective inhibitors of FAAH activity. Previous studies from our lab have identified a novel class of carbamate-based FAAH inhibitors, which are highly potent and selective. We will build on this knowledge to design and synthesize a second generation of carbamate-based FAAH inhibitors, and to investigate the molecular basis of their interaction with FAAH. These studies will set the stage for the molecular characterization of the anandamide transporter protein and for the development of agents that selective block anandamide transport or FAAH-catalyzed anandamide hydrolysis. In conclusion, by further elucidating the mechanisms of anandamide deactivation, our studies will shed new light on the mechanisms of marijuana abuse and help develop novel strategies for substance abuse and psychiatric disorders.